抗癌胚抗原单链抗体基因与Mn-SOD基因的融合及在昆虫细胞中的表达

将抗癌胚抗原单链抗体基因与锰 超氧化物歧化酶基因融合 (ScFv Mn SOD )插入昆虫杆状病毒供体质粒pFastBacHTb中 ,经大肠杆菌DH1 0Bac体内转座 ,产生重组杆状病毒pBacHTb Mn SOD ScFv。将其转染粉纹夜蛾Tn 5B1 4细胞 ,经扩增后在细胞内进行表达。SDS PAGE分析结果表明 ,融合基因得到高效表达 ,其表达产物相对分子质量为 40 0 0 0单体和 1 60 0 0 0左右的四聚体。Western印迹分析结果 ,以 6×His单克隆抗体为一抗进行蛋白印迹在相对分子质量 40 0 0 0单体和 1 60 0 0 0四聚体处可见表达条带 ,放射免疫分析表明重组杆状病毒表达ScFv Mn SOD融合蛋白能与CEA有较高的结合力 ,并且此融合蛋白具有特异SOD酶活性 ,酶比活可达 32 6 5u/mg。

Expression of the Fusion Gene of Manganese SOD Gene and Anti-Carcinoembryonic Antigen Single-Chain Antibody Gene in Insect Cells

The fusion gene encoding single chain antibody to carcinoembryonic antigen with human manganese SOD gene was inserted into the donor plasmids pFastBacHTb Then the recombinant plasmids were transformed to the E coli DH10Bac,in which transposition performed The recombinant bacmid DNAs,which contained the fusion gene,were extracted from the E coli DH10Bac,and were transfected the Tn 5B1 4 cells The recombinant bacularviruses were obtained After amplified,the recombinant bacularviruses infected the Tn cells and the fusion gene of ScFv Mn SOD was highly expressed in Tn cells SDS PAGE analysis revealed that the molecular weights of fusion gene were the monomer of 40 000 Mr and the tetramer of 160 000 Mr approximately Using anti 6xHis monoclonal antibody as the primary antibody in Western blots,both bands of 40 000 Mr and 160 000 Mr were detected And it was shown to possess the ability to bind to its specific antigen of CEA by RIA and had specific SOD activity